Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.16-10 ng/mL
Sensitivity:	0.09 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human SCFR/c-Kit in samples. No significant cross-reactivity or interference between Human SCFR/c-Kit and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human SCFR/c-Kit. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human SCFR/c-Kit and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human SCFR/c-Kit, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human SCFR/c-Kit. The concentration of Human SCFR/c-Kit in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	Kit: a receptor tyrosine kinase and a member of the subfamily that includes PDGF, CSF-1 and FLT-3/flk-2 receptors. Receptor for stem cell factor. Plays a critical role in hematopoietic stem cell, mast cell, melanocyte and germ cell development. Ligand binding induces autophosphorylation, dimerization and activation, leading to the recruitment and phosphorylation of downstream SH2-containing signaling components including PLC-gamma, PI3 kinase p85, SHP2 and CrkL, linking c-Kit to various cell signaling pathways. Molecular lesions that impair the kinase activity of c-Kit are associated with a variety of developmental disorders, while mutations that constitutively activate c-Kit can lead to hyperplasia and tumorigenesis. Activating mutations cause >90% of gastrointestinal stromal tumors (GIST); successfully treated with inhibitors Gleevec (imatinib, Glivec) and Sutent (Sutinib, SU11248). Activating mutations also induce mastocytosis. Autocrine/paracrine stimulation may drive some lung and other tumors. Loss of expression associated with melanoma progression. Familial loss of function mutations cause piebaldism, with defects in hair and skin pigmentation due to lack of melanocytes.
UniProt Protein Details:	Protein type:EC 2. 7. 10. 1; Protein kinase, TK; Protein kinase, tyrosine (receptor); Kinase, protein; Oncoprotein; Membrane protein, integral; TK group; PDGFR family Chromosomal Location of Human Ortholog: 4q12 Cellular Component: extracellular space; internal side of plasma membrane; mast cell granule; integral to membrane; acrosome; plasma membrane; intercellular junction; external side of plasma membrane Molecular Function:protein binding; protein homodimerization activity; protease binding; cytokine binding; metal ion binding; protein-tyrosine kinase activity; stem cell factor receptor activity; transmembrane receptor protein tyrosine kinase activity; receptor signaling protein tyrosine kinase activity; ATP binding Biological Process: nerve growth factor receptor signaling pathway; activation of MAPK activity; somatic stem cell maintenance; germ cell programmed cell death; positive regulation of JAK-STAT cascade; lymphoid progenitor cell differentiation; positive regulation of long-term neuronal synaptic plasticity; positive regulation of tyrosine phosphorylation of Stat3 protein; regulation of cell shape; epithelial cell proliferation; germ cell migration; somatic stem cell division; erythrocyte differentiation; T cell differentiation; fibroblast growth factor receptor signaling pathway; mast cell chemotaxis; embryonic hemopoiesis; stem cell differentiation; detection of mechanical stimulus involved in sensory perception of sound; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of tyrosine phosphorylation of Stat1 protein; immature B cell differentiation; regulation of pigmentation during development; glycosphingolipid metabolic process; positive regulation of transcription factor activity; mast cell cytokine production; peptidyl-tyrosine phosphorylation; protein amino acid autophosphorylation; signal transduction; ovarian follicle development; positive regulation of MAPKKK cascade; myeloid progenitor cell differentiation; melanocyte differentiation; positive regulation of cell proliferation; negative regulation of programmed cell death; visual learning; hemopoiesis; inflammatory response; positive regulation of Notch signaling pathway; lamellipodium biogenesis; epidermal growth factor receptor signaling pathway; phosphoinositide-mediated signaling; dendritic cell cytokine production; cytokine and chemokine mediated signaling pathway; male gonad development; stem cell maintenance; positive regulation of phosphoinositide 3-kinase activity; mast cell degranulation; regulation of cell proliferation; positive regulation of pseudopodium formation; positive regulation of tyrosine phosphorylation of Stat5 protein; gut development; pigmentation; actin cytoskeleton reorganization; innate immune response; spermatogenesis; spermatid development; positive regulation of cell migration Disease: Gastrointestinal Stromal Tumor; Mast Cell Disease; Piebald Trait; Testicular Germ Cell Tumor
NCBI Summary:	This gene encodes the human homolog of the proto-oncogene c-kit. C-kit was first identified as the cellular homolog of the feline sarcoma viral oncogene v-kit. This protein is a type 3 transmembrane receptor for MGF (mast cell growth factor, also known as stem cell factor). Mutations in this gene are associated with gastrointestinal stromal tumors, mast cell disease, acute myelogenous lukemia, and piebaldism. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P10721
NCBI GenInfo Identifier:	125472
NCBI Gene ID:	3815
NCBI Accession:	P10721. 1
UniProt Secondary Accession:	P10721,Q6IQ28, Q99662, Q9UM99, B5A956, D5LXN2, D5M931 F5H8F8,
UniProt Related Accession:	P10721
Molecular Weight:
NCBI Full Name:	Mast/stem cell growth factor receptor Kit
NCBI Synonym Full Names:	v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
NCBI Official Symbol:	KIT
NCBI Official Synonym Symbols:	PBT; SCFR; C-Kit; CD117
NCBI Protein Information:	mast/stem cell growth factor receptor Kit; p145 c-kit; proto-oncogene c-Kit; piebald trait protein; soluble KIT variant 1; tyrosine-protein kinase Kit; proto-oncogene tyrosine-protein kinase Kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene-like protein
UniProt Protein Name:	Mast/stem cell growth factor receptor Kit
UniProt Synonym Protein Names:	Piebald trait protein; PBT; Proto-oncogene c-Kit; Tyrosine-protein kinase Kit; p145 c-kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; CD_antigen: CD117
Protein Family:	Mast/stem cell growth factor receptor
UniProt Gene Name:	KIT
UniProt Entry Name:	KIT_HUMAN

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (ng/mL)	RLU	Average	Corrected
10	29795 35147	32471	32447
5	14391 16549	15470	15446
2.5	7820 7186	7503	7479
1.25	3626 3680	3653	3629
0.63	1776 1746	1761	1737
0.31	860 788	824	800
0.16	348 366	357	333
0	23 25	24	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human SCFR/c-Kit were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human SCFR/c-Kit were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.50	1.05	4.60	0.53	1.05	4.43
Standard deviation	0.05	0.07	0.37	0.06	0.12	0.37
C V (%)	10.00	6.67	8.04	11.32	11.43	8.35

Recovery

The recovery of Human SCFR/c-Kit spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	99-112	105
EDTA plasma (n=5)	95-112	103
Cell culture media (n=5)	94-109	100

Linearity

Samples were spiked with high concentrations of Human SCFR/c-Kit and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	96-108	96-111	96-111
1:2	Average (%)	101	101	101
1:4	Range (%)	101-118	101-113	90-102
1:4	Average (%)	109	106	95
1:8	Range (%)	98-114	98-111	87-102
1:8	Average (%)	106	104	94
1:16	Range (%)	95-111	88-98	98-109
1:16	Average (%)	102	93	103

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.